516 Intracellular Calcium-Mediated Oxidative Stress Is a Common Denominator in the Mechanism of Tight Junction Disruption by Different Types of Stress in CaCo2 Cell Monolayers

Abstract

Background: Matrix Metalloproteinase (MMP)-induced extracellular matrix remodeling modulates intestinal inflammation. MMP-12 is a human macrophage elastase capable of degrading basement membrane (BM). Defective intestinal barrier leading to increased intestinal permeability is an important pathogenic factor for intestinal inflammation. The role of MMP-12 in intestinal barrier function and intestinal inflammation remains unclear. We hypothesize that MMP-12 induces degradation of BM and helps macrophage transmigration thereby compromising intestinal barrier and augmenting intestinal inflammation. Aim: The aim of this study was to investigate the role of MMP-12 in intestinal epithelial tight junction (TJ) permeability and macrophage transmigration in experimental dextran sodium sulfate (DSS) colitis and in vitro Caco-2 cell model. Methods: Nine weeks-old wild type (WT) and MMP-12-/mice were administered 3% DSS in drinking water for 7 days. An in vivo colonic recycling perfusion and in vitro epithelial cell model was used to study epithelial permeability. Results: DSS treatment resulted in a marked increase in MMP-12 protein expression in WT colon. DSS administration significantly increased the colonic permeability in WT but not MMP-12-/mice (p<0.01). The loss of body weight, disease activity index, and histological lesion score of colitis was significantly attenuated in MMP-12-/DSS group compared to WT DSS group (p<0.01). In immunohistochemical study, the BM laminin was significantly lost in WT DSS colon but not in MMP-12-/DSS colon. The epithelial infiltration of macrophages in DSS colitis, as assessed by macrophage marker CD68 staining, was found to be significantly lower in MMP-12-/mice than WT mice. To further investigate the role of MMP-12 in intestinal TJ permeability, human intestinal epithelial Caco-2 cells were co-cultured with phorbol myristate acetate activated, MMP-12 secreting human macrophage U937 cells. The co-culture resulted into progressive and significant decrease in Caco-2 transepithelial resistance (TER) and concomitant increase in paracellular inulin flux, indicating loss of Caco-2 TJ barrier. Furthermore, siRNA-induced knock down of MMP-12 expression in U937 macrophage cells attenuated loss of Caco-2 TER and increase in inulin flux after U937 co-culture. Also, siRNA-induced knock down of MMP-12 significantly prevented U937 macrophage transmigration across the Caco-2 cells. Conclusion:The clinical severity, colonic permeability, and macrophage infiltration in colitis was attenuated in MMP-12-/mice. Macrophage derived MMP-12 increased intestinal epithelial permeability and enables macrophage transmigration in a cell culture model. These data suggest that MMP-12-induced macrophage transmigration and loss of intestinal epithelial TJ barrier contributes to the development of colitis.